ABSTRACT Blocking antibodies to CTLA4, PD-1 and other inhibitory surface receptors expressed on exhausted T cells, or blocking antibodies to the PD-1 ligands PD-L1 and PD-L2 expressed by tumor and stromal cells, have been remarkably successful at promoting long-term tumor regression; moreover combinations of blocking antibodies to multiple inhibitory receptors, often reinforced with activating antibodies to costimulatory receptors, have been more effective than treatment with individual blocking antibodies alone. Despite these successes, many patients still fail to respond to ?immune checkpoint blockade? therapies, emphasizing the need to understand immune cell ?exhaustion? at a molecular level, both in mouse models and in humans. The calcium- and calcineurin-regulated transcription factor NFAT is a driver of the transcriptional responses underlying T cell activation. The T cell activation program mainly depends on cooperative binding of NFAT and its transcriptional partner AP-1 (Fos-Jun) at composite DNA sites in gene promoters and enhancers. In parallel, NFAT can activate a second transcriptional program that imposes a hyporesponsive state, typically termed `exhaustion' or `dysfunction', that is observed in CD8+ T cells exposed to persistent antigen stimulation during chronic viral infections and cancer. This second program is characterized by a spectrum of functionally compromised states, including decreased cytokine expression and increased expression of multiple inhibitory receptors (PD-1, CTLA4, LAG3, TIM3, TIGIT). Thus an effective alternative to combination therapies might be to modulate the balance between NFAT-mediated programs of activation and exhaustion, so as to skew tumor- infiltrating T cells away from exhaustion and towards effector function. We will test this hypothesis here. Our experiments with an engineered NFAT1, minimally modified to prevent its interaction with AP1, have established that the transcriptional program of exhaustion is independent of the NFAT1-AP1 interaction. We have identified important targets of NFAT in the exhaustion program, including transcription factors of the Nr4a family. Moreover, we have shown that Nr4a transcription factors act in part by repressing the expression or activation of bZIP transcription factors that would otherwise promote an effector-like phenotype in exhausted tumor-infiltrating T cells. In Aim 1, we will identify and characterize these bZIP transcription factors; in Aim 2, we will define the differential roles of two NFAT family members, NFAT1 and NFAT2; in Aim 3, we will use novel proteomic strategies to identify NFAT-interacting proteins that help impose the exhaustion program; and in Aim 4, we will continue our successful search for small molecules that modulate the NFAT:AP-1 interaction. Our proposed studies will test the hypothesis that CD8+ TILs are functionally silenced by a cell-intrinsic transcriptional program mediated by NFAT activation and secondary repression of bZIP transcription factors. The results will contribute to a broad mechanistic understanding of the transcriptional mechanisms operating in mouse and human tumor-infiltrating T cells.